The overall goal of this procedure is to simultaneously assess multiple potential phosphorylation sites and to identify sites for subsequent validation by phosphorylation site mutants. This method can help answer key questions in the biochemistry and cell biology fields about the role of post-translational modifications in cell signaling. This technique allows easy budget friendly preliminary screening for phosphorylation sites and the protein of interest.
The implication of this technique extended toward the diagnosis and therapy of diseases including inflammation and cancer because phosphorylation is an important biochemical event in signaling pathways in human diseases. Aiding the demonstration would be Sol-Bi Shin a graduate student from my laboratory who while perform site directed mutagenesis. First combine 50 nanograms of the plasma pGEX JST NRF2, 125 nanograms of forward primer, 125 nanograms of reverse primer, one microliter of DNTP mix 2.5 units of PFU DNA polymerase and 10x reaction buffer on ice.
Add sterile distilled water to bring the volume to 50 microliters. Perform PCR to incorporate the mutant strand and then cool the sample on ice for two minutes. Add one microliter of DPNI restriction enzyme to the cooled sample.
Mix the sample with gentle pipetting and then centrifuge the sample for one minute at 13, 000 times G.Incubate the sample at 37 degree Celsius for two hours to digest the parental double stranded DNA. Then toss supercompetent E.coli cells with endA1 and RACA1 mutations on ice. Add 50 microliters of the reaction mixture to the chilled cells and incubate on ice for 30 minutes.
Meanwhile, preheat a heating block to 42 degree Celsius. Once incubation in complete, heat the mixture at 42 degree Celsius for 90 seconds and then cool the mixture on ice for two minutes. Add pre-warmed Lysogeny broth to the transformed cells.
Incubate the mixture at 37 degree Celsius for one hour while shaking at 180 RPM. Then spread the cells on LB ampicillin agar plates and incubate the plates overnight at 37 degree Celsius. Transfer a single colony to five milliliters of Lysogeny broth with ampicillin.
Incubate the samples overnight at 37 degree Celsius while shaking at 180 RPM. Extract the sample DNA using a standard mini prep kit for DNA sequence analysis. Run at least 200 nanograms of HDNA construct in a DNA analyzer to verify the mutant sequence.
Express and purify the validated mutant NRF2 protein. Determine the quantity of mutated NRF2 produced before proceeding the kinase assay. To begin the peptide competition assay, prepare a 5X kinase buffer solution with an adenosine monophosphate concentration of 500 micromolar.
Then thaw frozen samples of AMPK wild type human NRF2 and three oliogopeptides mimicking the AMPK phosphorylation sites of NRF2. For each peptide, while on ice, combine 0.43 milligrams of the peptide with 0.15 micrograms of AMPK, 0.4 micrograms of NRF2 and six microliters of 5X Kinase Buffer. Add sterile distilled water to achieve a final volume of 30 microliters.
Then in a shielded area, set a heating block to 30 degree Celsius. Add one microcurie of gamma 32P ATP to each tube, mixing each solution thoroughly by pipetting up and down. Incubate the reaction mixtures at 30 degree Celsius for 15 to 30 minutes.
During the incubation, prepare 7.5%STS PAGE gel. Then add three microliters of 10X STS sample buffer to each tube and mix thoroughly to stop the kinase reaction. Run the samples in the gel at 70 volts for 20 minutes and then at 140 volts for one hour.
Upon completing the gel electrophoresis carefully remove the highly radioactive gel below the bromophenol blue line. Gently transfer the gel from the caster to a glass tube. Fix the gel in a solution of 50%methanol, 40%water and 10%acetic acid for 20 minutes.
Then gently place the gel on filter paper and cover it with transparent wrap. Dry the gel with a vacuum gel dryer for one hour at 80 degree Celsius. Exposure the dry gel either to a phosphorus screen or an x-ray film for approximately 16 hours for two days respectively.
Use a Phosphorimager to generate a high resolution TIFF or Bitmap file of the screen. To begin the AMPK activity assay thaw samples of AMPK and of mutant and wild type GST NRF2 on ice. Combine 0.4 micrograms of mutant GST NRF2 with 0.15 micrograms of AMPK and six microliters of 5X Kinase buffer.
Add sterile distilled water to bring the volume to 30 microliters. Prepare another reaction mixture in this way with 0.4 micrograms of wild type GST NRF2. Add one microcurie of gamma 32P ATP to each reaction vial.
Pipette the mixtures up and down several times and then briefly centrifuge the mixtures. Incubate the mixtures at 30 degree Celsius for 30 minutes in a shielded area and then terminate the kinase reaction with three microliters of 10X STS sample buffer. Run STS PAGE, fix the gel, and dry the gel using the same conditions as the competitive kinase assay.
Expose the gel to a phosphorus screen overnight and scan the screen with the Phosphorimager. An in vitro AMPK assay was performed in the presence of three 10 residue oligopeptides mimicking AMPK phosphorylation sites on human NRF2. The oligopeptide mimicking the Serine 558 site offered the greatest competition for AMPK phosphorylation.
An NRF2 mutant replacing Serine 558 with alanine was then synthesized and evaluated in an in vitro activity assay. Very little AMPK phosphorylation of the S558 A mutant was observed indicating that AMPK directly phosphorylates human NRF2 at Serine 558. Since peptides competitively bind to proteins the principle of this method may also be applied to identifying other post translation and modifications such as acetylization and lysine residues and protein protein interactions.
Don't forget that working with radio isotope can be extremely hazardous. Radio isotope safety training should already be taken before performing this procedure.
